Dynamic Light Scattering (DLS)

 
 

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Instrument:


The Zetasizer Auto Plate Sampler is located in the Michael Swann building, room 3.10.


 

Please click here for individual platform/resource costings.

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Features of the Zetasizer APS instrument:

  1. The size range of molecules (radius) detectable is 0.3 nm (corresponding to ca 380 Da protein) to 0.5 microns.


  1. Dual temperature control allows for separate temperatures to be set for the plate compartment (5 °C to 40 °C) and for the measurement cell (2 °C to 90 °C)


  1. Direct and label-free measurement in solution, of binding affinity, stoichiometry, enthalpy and entropy.


  1. The software uses a graphical user interface for setup and results display.


  1. 96- and 384-well plate format, with a level sensor which simplifies the measurement set-up.


  1. The sample is recoverable after measurement.


  1. Recommendations for good reproducible size measurements for solutions of a "typical" protein of 15-25 kDa are:

    1. minimum volumes of 45 μl (384-well) or 70 μl (96-well) per sample.

    2. concentrations of ≥ 1.5 mg.ml-1.

    3. higher sensitivity is achievable with larger proteins and aggregation is detectable in protein solutions ≥ 0.2 mg.ml-1.


Download technical information on the Zetasizer APS here

For more information from Malvern Instruments Ltd, go to http://www.malvern.com and for information on light scattering technology applied to protein characterisation, go to http://www.malvern.com/labeng/industry/protein/proteins.htm

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Biological applications of Dynamic Light Scattering

DLS is a rapid, non-invasive technique to determine the hydrodynamic size of macromolecules in solution and/or to provide size distribution profiles of mixtures of molecules.

DLS is an ideal technique for screening samples, e.g. to characterize proteins in a variety of solution conditions and obtain a profile of their state in solution. It can quickly tell you if your assay conditions are compatible with your molecules of interest. Thus DLS should be employed early in your research in order to determine solution conditions which:

  1. provide the biologically relevant oligomeric state/quaternary structures

  2. ensure the sample is monodisperse

  3. provide the biologically relevant oligomeric state/quaternary structures.

  4. ensure optimal stability

  5. induce changes in quaternary structure

  6. promote/disrupt intermolecular complex formation

  7. induce/minimise aggregation

  8. are optimal for protein crystallizatio


DLS can also measure:

  1. the thermal melting point of macromolecules (results are comparable with that obtained using the thermal denature assay on the Biorad IQ5 ICycler)

  2. estimate the molecular weight of proteins

  3. provide indication of shape of protein if molecular weight is known.


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What is Dynamic Light Scattering?

Dynamic Light Scattering (DLS) is also known as Photon Correlation Spectroscopy (PCS) and Quasi-elastic Light Scattering (QELS).

In DLS, a laser beam is directed into a solution and, when it meets a particle, the light energy is scattered in all directions; the scattered light is detected on a photon detector connected to a correlator. The scattered light is a function of the hydrodynamic size and the concentration of the sample. Higher concentrations of molecules scatter more light than lower; larger molecules scatter more light than smaller molecules at the same concentration. Time-dependent fluctuations in the scattered light occur because the particles are moving under Brownian motion; these fluctuations are directly related to the rate of diffusion of the molecules in the solution. The correlator compares the intensity of the scattered light at time t with that at time t+δt, t+2δt, t+3δt, etc, and thus generates a correlation function. Since small molecules diffuse faster, they lead to a faster decay in the measured correlation function. The measured decay rate is thus related to the hydrodynamic size of the scattering object.


The correlation function generated is then fitted with:

  1. a single exponential to obtain the mean diameter (termed the Z-average) and an estimate of width of the distribution (i.e. the polydispersity).

  2. a multiple exponential to obtain the distribution of particle sizes.



Measurements of hydrodynamic diameters of proteins by dynamic light scattering in the BCF show excellent agreement with those determined by gel filtration chromatography in the PPF, as shown below for protein standards analysed on the Zetasizer APS (y axis) and on a GE Healthcare Superdex 200 column (x-axis).


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Booking and rules for use of the Zetasizer APS:

The machine must be booked prior to use.  A web-based booking system is available for registered users at the link below. You cannot book more than 4 weeks in advance. A half day, a single individual day or up to five consecutive days can be booked at any one time. You cannot book two sets of five consecutive days in consecutive weeks, without prior agreement with the EPPF staff. 


Click here for DLS online booking


24 hours’ notice should be given if a booked slot cannot be used.  If the machine is booked and not used, the booked user will be charged a nominal fee (click here for details).  The system administrators can delete booked user slots with 24 hours’ notice.


Please note that if you are using the machine and it is damaged or misused the PI responsible for the user is liable for the cost of repairs.  If you don’t know what you are doing, do not attempt to use the machine, please speak to Liz Blackburn, Martin Wear, Matt Nowicki, or Sandra Bruce.

 

contacts:


Facilities Manager:

Dr. Martin Wear

Rm. Swann 3.20

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: martin.wear@ed.ac.uk


Lab Manager:

Mrs. Sandra Bruce

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: sabruce@staffmail.ed.ac.uk


Protein Technologist:

Dr. Liz Blackburn

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: E.A.Blackburn@ed.ac.uk


Protein Technologist:

Dr. Matt Nowicki

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: matthew.nowicki@ed.ac.uk



Facilities:


CTCB-Home

Protein Production Biophysical Characterisation

In Silico Screening

Training


Links:


On-line Booking

Core Column Library


Access Charges/Costs:


Access Charges/Costs

 

Location:

The University of Edinburgh,

Level 3 Michael Swann Building,

King’s Buildings,

Mayfield Rd.,

EH9 3JR,

UK


Lab 3.10

Zetasizer APS - DLS